03 Mar 2023
by Richa

Summary- paper 22: Cryo-EM structures of human SPCA1a reveal the mechanism of Ca2+/Mn2+ transport into the Golgi apparatus

Zhenghao Chen, Satoshi Watanabe, Hironori Hashida, Michio Inoue, Yasukazu Daigaku, Masahide Kikkawa, Kenji Inaba

Science Advances, 2023

Questions/gaps addressed:

  • The mechanism of Ca2+ homeostasis in the Golgi apparatus which also acts as an intracellular calcium storage compartment, is less well understood than that in the ER. SERCA (Sarco(endo)plasmic Ca2+–ATPase) localizes to the ER and cis-Golgi, SPCA1 (Ca2+/Mn2+-ATPase) is present in the trans-Golgi and TGN for Ca2+ uptake, generating a large Ca2+ gradient concentration between the ER (~500 μM) and Golgi (~100 μM). How are SERCA and SPCA1 activities regulated?

  • Loss-of-function mutations in the ATP2C1 (gene encoding SPCA1) causes an inherited autosomal dominant disease, Hailey-Hailey disease, a skin disorder characterized by persistent blisters. Homozygous KO of the ATP2C1 gene in mice is embryonic lethal due to failed neural tube closure. What is the structural basis of the defects in keratocytes and neuroepithelial cells in SPCA1 LOF?

  • SPCA1 transports both Mn2+ and Ca2+ into the Golgi lumen, with a higher affinity for the former. Mn2+ is a crucial cofactor for many enzymes, and too much can also be toxic. Structural basis for difference in Mn2+ and Ca2+ transport by SPCA1?

Key methods:

  • PA-tag (GVAMPGAEDDVV) and a tobacco etch virus (TEV) cleavage site at the N terminus of SPCA1 isoform 1a, expressed in HEK293T using the PiggyBac Cumate Switch Inducible system, induced with cumate (150 μg/ml) and 50 nM phorbol 12-myristate 13-acetate (PMA) for overexpression, solubilization in 1% DDM, and purification with anti–PA-tag Antibody Beads.

  • SPCA1a is ubiquitylated at Lys8 (A-domain) and Lys496 (N-domain). The purified protein was deubiquitinated by treatment with Usp2cc enzyme to prepare a homogeneously deubiquitinated SPCA1a sample for cryo-EM.

  • EnzCheck phosphate assay to monitor in vitro ATPase (and by proxy transport activity) of purified SPCA1. ATPase activity observed with multiple divalent metal cations- Co2+ and Ni2+, as well as with Ca2+ and Mn2+.

  • To increase density of the A-domain of SPCA1, tested human cofilin-1 (hCFL-1) as a binder protein since it is known to bind the P-domain of SPCA1a but observed no interaction between purified hCFL-1 and SPCA1a in vitro assay using size exclusion chromatography. Instead focussed on stabilization by binding to a nanobody

  • Flow cytometry screen for a nanobody that binds specifically and tightly to SPCA1a using a previously published yeast surface-displayed nanobody library (McMohan et al 2018, NSMB), found Nb14 to bind the N-domain of SPCA1a. Converted Nb14 to a megabody by fusing a scaffold protein to Nb14 while retaining the binding affinity for SPCA1a.

  • To compare TM helix interactions between SERCA1a, SERCA2b, and SPCA1a, counted the number of H-bonds formed between different TM helices.

Major takeaways:

  • The physiological significance of SPCA1a ubiquitylation is unclear.

  • cryo-EM structures of human SPCA1a in two intermediate states at 3.1- to 3.3-Å resolution:
    • the E1-ATP state using β,γ-methyleneadenosine 5′-triphosphate (AMPPCP), a nonhydrolyzable analog of ATP, in the presence of Ca2+ or Mn2+, and
    • the E2P state using BeF3−, a phosphate group mimic, in the absence of these metal ions.
  • SPCA1a shares the typical architecture of P-type ATPases, with 10 TM helices (TM1 to TM10) and three cytosolic domains, including the actuator (A), nucleotide-binding (N), and phosphorylation (P) domains. The TM domain of SPCA1a consists of three TM helix clusters (TM1–2, TM3–4, and TM5–10), similar to SERCA.

  • The overall arrangement of the cytosolic and TM domains in SPCA1a resembles that in SERCA and that the location and amino acid residue geometry of the Ca2+/Mn2+-binding site in SPCA1a are nearly superimposable with those of the second Ca2+-binding site (site II) in SERCA2b.

  • While the cytosolic domain arrangement is similar between SPCA1a and SERCA, the mode of interactions at the cytosolic domain interfaces is different between these two Ca2+ pumps. The Ca2+/Mn2+-binding site is primarily constituted by TM4 and TM6. Mn2+ occupies the same binding site in SPCA1a as Ca2+.

  • Difference between SPCA1a and SERCA2b in TM2. TM2 is directly linked to the A-domain, the observed twist around the cytosolic end of TM2 in SPCA1a suggests that TM2 is one of the key elements that modulate the metal ion transport by this Ca2+/Mn2+ ATPase. TM2 twisting seems likely to be an essential event of several metal transporting P-type ATPases (SPCA1a, SERCA1, ATP13A, lipid P4-ATPase). Larger conformational and positional flexibility of TM2 and TM6 in SPCA1a compared with SERCA may help to explain the wider metal selectivity of SPCA1a.

  • Appears that smaller numbers of H-bonds are formed between TM6 and its neighboring TM helices in SPCA1a (5 interdomain H-bonds with neighboring TMs), compared to those in SERCA1a and SERCA2b (10 and 11 H-bonds).

  • The mode of ATP binding in SPCA1a is highly similar to that in SERCA2b.

  • Hailey-Hailey disease associated mutations: most mutations occur around the metal-binding pocket and in the P-domain, suggesting that these mutations affect the metal transport activity, efficiency of ATP hydrolysis, and/or phosphorylation of Asp350 of SPCA1a.

  • The Q747A gain-of-function mutation enhanced the SPCA1-mediated uptake of Mn2+, while the corresponding Q783A mutation in yeast PMR1 blocked Mn2+ transport. Why?